CA2150471A1 - Megakaryocyte and platelet growth, production and composition - Google Patents

Abstract

Megakaryocytes and platelets are produced in culture by employing conditions inhibiting the formation of fibroblasts. Particularly, heparin is found to inhibit fibroblasts as well as inactivating factors produced by platelets which inhibit megakaryocyte formation. Isolation of a CD34+41+ cell population which enriches for both megakaryocyte and granulocyte-macrophage progenitors is provided.

Description

~O 94/13306 215 n ~ 71 PCT/US93/11870 MR~R~O~. y-l & AND PI~AT&LET GROWTlI, PROL~ ON AND COMPOSITION

INTRODUCTIQN
T~chnical Field The field of this invention i5 growth and production of hematopoietic cells, more particularly megakaryocytes and platelets, and purification of a human megakaryocyte and granulocyte-macrophage progenitor.

~ackqround Thrombopoiesis i8 the differentiation and maturation of megakaryocytes and platelets. Despite enormous efforts that have been made to understand the process of thrombopoiesis and the factors involved with thrombopoiesis, there rem~; n-~ an extraordinary amount of confusion and uncertainty about the process. Fac~ors that appear to have effects in vitro seem to have little or no effect in vivo. An activity called thrombopoietin has been elusive and has yet to be i~olated in pure form and characterized. Efforts to provide for long-term culture of megakaryocyte progenitors providing for their maturation and formation of platelets has also been problematical.

W094/~306 l PCT~S93/11870 ~

2 ~ 7 1 2 Thrombopoiesis is an extremely important process for the health of individuals. Platelets play a vital role in a number of protective processes, providing various factors, participating in clotting, and the like. There is, therefore, substantial interest in providing procedures which will aid in the understanding of thrombopoiesis, as well as providing for long-term production of megakaryocytes and platelets which may find application as therapeutic procedures.
Relevant Literature Volume 15, number 1, 1989 of Blood Cells is dedicated to megakaryopoiesis. Hill and Levin, Blood Cells (1989) 15:141-166 describe regulators of thrombopoiesis. A
discussion of the role of heparin with endothelial cells may be found in Thornton et al., Science (1983) 222:623-5.
WO 90 US1725 reports a megakaryocyte growth promoting activator factor protein. Regulation of megakaryocyte differentiation by thrombin glycosaminoglycans has been reported.

SUMMARY OF THE INVENTION
Methods are provided for growing megakaryocytes and platelets in culture for extended periods of time, by employing media which encourage megakaryocyte progenitor and megakaryocyte growth. Various sources of megakaryocyte progenitor cells are employed. A plasma source of megakaryocyte growth factor is provided for enhancing megakaryocyte progenitor and megakaryocyte growth. Assay systems are provided for assaying for the effect of factors on the proliferation and maturation of megakaryocyte progenitors and megakaryocytes. The megakaryocytes and platelets find use in the treatment of thrombocytopenia, identification of megakaryopoiesis activity of compounds or compositions, and for understanding the mechanism of megakaryopoiesis.

~W094/~306 215 0 4 7 1 PCT~93tll870 DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for the isolation and growth of megakaryocyte progenitors, megakaryocytes and platelets, in culture and in vivo.
Megakaryocytes can be grown in the presence of heparin and a megakaryocytic growth factor. In culture, the megakaryocytes may be maintained for extended periods of time without replenishment of the progenitor cells from an exogenous source.
Bioassays are provided for monitoring megakaryopoiesis and factors influencing megakaryopoiesis.
The megakaryocyte progenitors, megakaryocytes and platelets can be produced by growing in culture, in an appropriate nutrient medium, human progenitor cells under conditions which are not supportive, preferably are inhibitory, of fibroblast growth, while inactivating inhibitors of megakaryopoiesis, particularly those produced by platelets. Megakaryocyte cultures may be established with cells from any convenient source, including bone marrow, fetal liver, non-adherent cells from bone marrow, fractionated cells, leukocytes, e.g., buffy coat free of erythrocytes, CD34+ fraction from bone marrow, low density cells, or the like, so long as early progenitors are present which are capable of maintaining production of megakaryocytes.
For any long-term culture, the cell composition which is employed will desirably include early hematopoietic progenitor cells, e.g. stem cells, lineage committed cells, or megakaryocyte progenitors. The cellular composition may be fetal, neonatal, or adult. While the subject methodology may be used with any primate hematopoietic cellular composition comprising early progenitor cells, the methodology desirably finds use with human cell compositions.
For many purposes, the growth of megakaryocytes, megakaryocyte progenitors, and platelets is desirable.
Thus, having a system whereby one can grow cells of the megakaryocytic lineage is extremely important in providing W094l~306 , PCT~S93/11870 ~
~l~Q471 4 a source of megakaryocytic cells and platelets. The following describes the method for growing cells of the megakaryocytic lineage and platelets in culture.
For the most part, containers which are used for tissue culture are coated to enhance fibroblast formation.
In accordance with the subject invention, however, containers are employed which remain uncoated or have been treated to avoid encouraging fibroblast formation.
Containers can be obtained where the containers are free of coating, either by being commercially available or by requesting such containers from sources such as Falcon, Corning, or the like. The containers may include multiple well plates, roller bottles, fermentors, Petri dishes or the like.
The medium which is employed may be any convenient medium providing the required salts, minerals and-nutritional supplements, such as amino acids, glucose and vitamins. Also, a small amount of a physiologically acceptable reductant is used such as 2-mercaptoethanol.
The mercaptoethanol will generally be present in from about 10-4 - 10-6 M, preferably about 10-5 M.
Various additives may be employed to discourage or inhibit fibroblast formation and proliferation. Additives which may be employed include heparin, citrate, or other additives that prevent aggregation and degranulation of platelets. The amount of heparin will be at least about 50 ~g/ml, preferably at least about l00 ~g/ml and usually not exceed 4 mg/ml, more usually not exceed 2 mg/ml, preferably being in the range of about 0.5 to l.5 mg/ml.
The amount of citrate will vary, when present, generally in the range of about 0.l to 1%. Other additives will be used in accordance with their activity and any adverse effects on the system. The medium generally will also have a low level of human plasma or serum, preferably using plasma at a concentration of about 10% or less, more preferably being about 5%, there generally being at least about 1% in the absence of specific cytokines in a defined medium. The amount of plasma, as well as the other ~ro 94/L3306 2 1 5 0 4 7 1 PCT/USg3/ll870 additives, may be optimized, where cells of the megakaryocytic lineage are encouraged to grow, while fibroblast growth and proliferation may be inhibited. The use of plasma is preferred, as serum contains factors which may inhibit the growth of megakaryocytes.
Any complex eukaryotic growth media may be used, which includes MCDB107, IMDM, RPMI, EX-CEL, EX-VIVO, etc., normally enhanced with a low level of plasma.
Desirably, factors which may be produced by platelets and inhibit megakaryopoiesis will be maintained at a low level. Factors of particular concern include platelet factor 4 (PF-4) and its degradation product thrombospondin. It is found that heparin is able to bind to PF-4 and diminish its inhibitory activity against megakaryopoiesis. Thus, when heparin is maintained in the medium, the heparin prevents the PF-4 and its degradation product from exerting their inhibitory influence. In the absence of heparin, other methods may be used for minimizing the level of PF-4. This can be achieved by continuous changing of the medium and removing PF-4 and its degradation products from the medium, the addition of antibodies to PF-4, dialysis for removal of PF-4, and the like.
One may control the calcium level and the calcium/phosphate ratio to enhance the preservation of platelets which are formed in the culture. Generally the calcium level should be in the range of about 0 to 10mM, while the phosphate concentration will be in the range of about 0 to 10mM. The level of ADP may also be controlled by providing for a system to convert ADP to ATP. A
convenient system includes creatinine phosphate and creatinine phosphate kinase, although other systems may find use. The concentration of the creatinine kinase and creatinine kinase phosphate will be maintained in a range to provide the desired ADP/ATP levels. These concentrations are not critical and are intended to be primarily of convenience, enhancing the productivity of W094/~3~ PCT~S93/11870 _ 215~L i 6 the system. Platelet preservation may also be achieved with dextrose acid citrate.
Defined growth factors may be added to the cultures in combination with megakaryocytic specific factors.
Growth factors include the interleukins IL-l, -3, -6, GM-CSF and erythropoietin, particularly at relatively high concentrations, usually greater than 25 ng/ml, preferably greater than 50 ng/ml, and may be lO0 ng/ml or higher.
Megakaryocytic specific growth factor is obtained from human plasma by fractionation.
The cell composition obtained in the adherent layer of the subject cultures is highly enriched for megakaryocytes. Greater than 10% of adherent cells by number, usually greater than 50%, are CD41+. Megakaryocyte progenitors are present in the liquid media, as non-adherent cells can be transferred to a new dish to initiate a secondary culture enriched for megakaryocytes.
A culture is obtained comprising non-adherent progenitor cells, adherent cells committed to the megakaryocyte/platelet lineage, and an adherent population of stromal/macrophage cells. The conditioned medium is supportive of megakaryopoiesis by virtue of comprising the appropriate growth factors, agent(s) for stabilizing platelets and agent(s) for inhibiting fibroblast growth and proliferation. Factor(s) present in plasma which are associated with megakaryopoiesis are found to be stable at 56C for at least 30 minutes.
In carrying out the production of megakaryocytes and platelets, usually from about lX105 to lX107 human bone marrow cells per ml will be seeded, preferably lxlO6 cells per ml, or a unit of leukocytes (buffy coat) from an adult, containing about 2 x lO8 white cells and freed of erythrocytes.
Cultures may also be initiated with megakaryocyte progenitor cells, preferably isolated away from other hematopoietic cells. A progenitor cell which is capable of initiating megakaryopoiesis in culture has the surface phenotype of CD41+, CD34+ and CDl5-. Progenitor cells may ~ W094/~3~ 215 n ~ 7 1 PCT~S93/11870 be isolated from fetal or adult bone marrow, which is obtained from a source of bone, eg. tibiae, femora, spine or other bone cavities. Other sources of human progenitor cells include embryonic yolk sac, fetal liver, fetal and adult spleen, and cord blood.
Of particular interest is the use of apheresed blood cells from patients treated with G-CSF, particularly in conjunction with chemotherapy, as a source of CD34+41+
progenitors. A relatively high percent of CD34+ cells in apheresed blood express CD41.
In some populations, e.g. fetal bone marrow, the cells which express CD34 may be split into distinct populations which are CD34~h and CD341~. Both populations stain with anti-CD34 antibodies at a higher level than they stain with an isotype matched control antibody.
While CD41+ cells are present in both the CD34h~b and CD34~ populations, the percentage of CD41+ is higher among CD34~h cells and the activity for culture initiation was higher in the CD34~h population.
Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, eg. complement and cytotoxins, and "panning"
with antibody attached to a solid matrix, eg. plate, or other convenient techn;que. Techniques which provide accurate separation include the use of fluorescence activated cell sorters. The isolated cells will be a substantially pure progenitor cell population, preferably at least 80% pure, more preferably at least 90%, and most preferably at least 95% pure.
Conventional physiologic conditions will be employed for growing human cells in culture, namely about 37C, in a humidified chamber, with air having about 5% carbon dioxide, where the oxygen will be at least about 5%
usually at least about 20%. Conveniently, wells of 6 - 10 cm or 96 well plates may be employed, which allows for the growth of the adherent cells to the surface. Within about W094/~3~ PCT~S93/11870 ~

7 days, megakaryocytic cells can be observed and the culture may be contihued for at least one month. Unless dialysis is used, the media is changed on a regular basis, generally at least about twice weekly, preferably at least about every 2 days.
One may observe islands of cells, with adherent cells set down, which differentiate to mature megakaryocytes, observed as multi-nucleated cells. The observed adherent layer is a combination of megakaryocytes, macrophages and stromal cells. The megakaryocyte cells are shown to have the surface protein markers CD41 (IIb/IIIa) (integrin) and CD42 (Ib), and when the cells are stained with Wright Giemsa, show the multinucleated or polylobulated histology characteristic of megakaryocytes. The progenitor cells, by comparison, will carry the CD41 and CD34 markers and be mononucleated.
Megakaryocytic cells may be further distinguished by staining with Megacolor (Cytocolor, Inc., Hinckley, OH).
In megakaryocytes, the cytoplasm stains intensely purple.
Megacolor staining may be performed on whole cells for histologic examination. For quantitative assays, the cells are lysed after staining, and the absorbance of the lysate read at A5~. Agar plates may be dried down and stained with Megacolor to assay for the number of megakaryocytic colonies present. In this way, limiting dilution plates can be read for the number of megakaryocytic progenitors present when the cultures were set up.
Alternatively, the level of src protein expression and its kinase activity may be used as a measure of megakaryocytic cell growth, measuring megakaryocyte progenitors, megakaryocytes and platelets. High level src expression is characteristic of platelets and megakaryocytes. The assay can be carried out with cellular lysates of relatively homogeneous or heterogeneous phenotype or populations selected by one or more markers, isolating the src kinase, e.g. by means of specific antibodies, and then measuring src kinase ~ 094/~306 21~ O ~ 7 1 PCT~S93/11870 activity under conditions supportive of src kinase catalyzed phosphorylation. To remove non-specific proteins from the isolated src kinase, one or more washes may be employed. By using a ~abeled nucleotide triphosphate, e.g. radioisotope labeled, one can determine the level of phosphorylation as indicative of the presence of cells associated with megakaryopoiesis. Alternatively, Western blotting can be used to quantitate src protein.
As the cultures mature, platelets accumulate.
Platelet concentrations of lO5-lO6/ml have been counted on an automatic cell counter. Platelet-like particles have also been identified by scanning electron microscopy. The platelets observed on processes of adherent cells are usually from 1-3 ~m in diameter.
The cells may be harvested by separating the adherent layer mechanically, using chelating agents, e.g., EDTA, sonication, enzymes, e.g., proteases, such as trypsin, collagenase, etc., or the like. The particular manner employed is not critical to this invention. The megakaryoblasts and megakaryocytes may then be isolated using various ter-hn;ques, such as cytospin, FACS, affinity separation, density separation, magnetic bead separation, and the like. As a result, a substantially pure cellular composition can be obtained of cells dedicated to megakaryopoiesis, namely dedicated megakaryocyte progenitors, megakaryocytes and platelets. The composition will be free of components of peripheral blood, which might be associated with isolation of such cellular composition from bone marrow or blood. In addition, each of the types of cells may be isolated in substantially pure form, namely, megakaryoblasts, megakaryocytes and platelets, generally comprising at least 75% by number of the same cellular type, usually at least about 90% of the same cellular type and up to and including about 100%.
Plasma may be used for isolation of the factors associated with megakaryopoiesis. In a preferred embodiment, affinity chromatography is used to deplete WOg4/~3~ PCT~S93/11870 ~
2 1 ~ o albumin. Ion exchange chromatography, such as DEAE, is then used to select for negatively charged proteins. In an elution with a NaCl gradient, typically from 0 to 0.5 M
NaCl, the megakaryocyte activity elutes at about O.lM to 0.3 M NaCl. Isoelectric focusing and sizing may be used for further enrichment.
In an alternative embodiment, fractionating ammonium t sulfate precipitation may be used to enrich protein fractions for growth factors. Further enrichment is achieved using gel permeation to separate different size fractions, followed by assaying for the enriched fractions. The enriched fractions are then further enriched using rp-HPLC, eluting with an aqueous acidic medium against n-propanol or acetonitrile, e.g., 0.1%
trifluoroacetic acid, or l M acetic acid. The fractions are bioassayed and enriched fractions may be further purified by gel electrophoresis or other separation technique.
A megakaryocytic growth factor can be obtained by fractionating medium from the culture or human plasma through a heparin column. The factor is characterized by binding to heparin, capable of being transferred from heparin-Sepharose to heparin in a conventional comple~
eukaryotic growth media, with heparin at a concentration of at least about l mg/ml, preferably at least about 3 mg/ml, and at 4 C. The ratio of heparin-Sepharose beads to medium will generally be at least about l g:2 ml, preferably 5 ml, and not more than about l g:lO ml. The factor is further characterized by supporting cell growth and proliferation of cells of the megakaryocytic lineage in the culture system as described above, while its absence results in the substantial absence of proliferation and maturation of cells of the megakaryocytic lineage.
The subject factor may be further purified by conventional techniques as described above to provide for l,000-fold or greater enhancement in concentration and specific activity.

~ 094/~3~ 2 1 ~ O 171 PCT~S93/11870 In order to evaluate the response of megakaryocyte progenitors to various factors, the factors may be evaluated using a standard semi-solid medium assay composition, e.g., methylcellulose assay composition comprising factors which support megakaryocyte growth. The methylcellulose which is employed will generally be at least about 0.8% and usually not more than about 1.5%
weight/v in water. The cellular source may be the same as that used for the culture. Also included in the culture will be at least about 15% fetal calf serum, fetal bovine serum, or human plasma, preferably about 20%. A
convenient volume for carrying out the assay is to employ about 200 ~l in a 24-well plate.
To support growth of the megakaryocytes, high concentrations of the cytokines IL-1, -3, -6 or GM-CSF, as well as erythropoietin are included, generally at least at about 0.5 times saturation or higher. Other factors may be included such as sl or steel factor at comparable concentration. By saturation it is intended that a further increase in concentration does not have a significant effect on the number of colonies of cells which are produced. Conveniently, about 10 ng/ml of each of the factors may be employed.
The cells which are seeded will include CD41+, CD34+
cells, which cells serve as megakaryocyte progenitors.
Granulocytes and macrophages may also be produced from these progenitor cells. The cells may be whole bone marrow or an enriched fraction comprising CD41+, CD34+
cells. The mature cells in the colonies which form will, for the most part, be free of the CD34 marker and will normally have the CD41 marker, as well as having the morphology of the mature megakaryocyte.
~ Agar may also be used as a semi-solid medium for colony assays. Such medium will contain bacto-agar at a - 35 concentration from 0.2 to 0.5% weight/volume in any standard culture medium. The medium will include human platelet poor plasma at a concentration of 20-30% v/v.
Growth factors such as IL-l, IL-3, IL-6, IL-11, GM-CSF and W094/~306 PCT~S93/11870 ~
215~471 12 erythropoietin, as well as PMA may also be included at saturating concentrations. Agar colonies have the advantage that the media may be dried down, and the colonies stained for t'hè presence of megakaryocyte-specific cell surface markers, e.g. CD41, or with Megacolor.
A factor or composition of interest may be studied by adding such factor or composition to the medium with all of the above-indicated factors or where one or more of the above-indicated factors are lacking. Thus, one may determine the inherent capability of a composition in conjunction with human plasma to stimulate megakaryocyte growth or its ability to interact with other factors or cytokines to provide for megakaryocyte growth. The CFU-mega assay is carried out for at least about 14 days,usually not more than about 18 days, preferably from about 14 to 16 days. The BFU-mega assay is carried out for at least 21 days, not more than 29 days, usually about 28 days. The conditions for the assay are that the liquid medium, methylcellulose or agar, is maintained at 37 C, 5%
Co2 in a humidified chamber.
By employing various dosages of a composition or factor, one can determine the activity of such composition or factor. In this manner, screening can be carried out to determine whether a particular composition or factor is useful for the growth of megakaryocytes or the production of platelets.
The megakaryocyte progenitors and/or megakaryocytes may be used for the treatment of thrombocytopenia by themselves or in conjunction with the infusion of platelets. In many instances, such as surgery, chemotherapy, etc., autologous megakaryocytic cells may be expanded in culture and returned to the host to minimize bleeding or other effect of the treatment which megakaryocytes and/or platelets may address. The cells may be infused in a physiologically acceptable medium, e.g., saline, generally being present at a concentration of at least about lO5 cells/ml, usually in the range of ~ W094/~306 21~ O ~ 71 PCT~S93/11870 about lo6 - lo8 cells/ml. The dosage will generally vary in the range of about 1O6 _ 1O8 cells/kg of host. Usually, the composition will be at least 70%, usually at least 90%
by weight of megakaryocyte progenitors and megakaryocytes.
One or more infusion may be necessary, as required.
Platelets may be harvested and administered in accordance with conventional ways.
An important cell for cellular therapy is a progenitor which can quickly give rise to granulocytes and platelets in vivo to hasten recovery of bone marrow post transplantation. A cell has been identified in fetal bone marrow which is CD34+41+ and has a high proliferative potential, giving rise to, at least, granulocytes, macrophages and megakaryocytes. The CD34+41+ cell from fetal bone marrow is a multipotential early progenitor.
Such a cell is ideal for cellular therapy, to provide fast engraftment of granulocytes and platelets.
The megakaryocytic cells may be used for gene therapy, where the cells may serve to maintain function under conditions where normal megakaryocytic cells may be adversely affected or to provide a function normally-unavailable from megakaryocytic cells. For example, the multiple drug resistance (mdr) phenotype may be introduced into the cells, which would make them resistant to chemotherapeutic agents, allowing for the continued normal megakaryocytic response during the chemotherapy treatment.
Genes encoding growth factors may be introduced, which would provide an enhanced source of growth factors in the bone marrow, where a deficiency might exist during a particular episode or one wished to enhance the growth of megakaryocytes or other hematopoietic cells. Various methods are known for providing transformation of cells, providing for stable extrachromosomal elements or homologous or random integration. Viral vectors incapable of replication have been described in the literature, which can be used for infection, transfection and integration. By being able to grow megakaryocytic cells WOg4/~3~ PCT~S93/11870 21~Q~71 14 in culture, one has the opportunity to transform such cells and P~pAn~ and clone desirable transformants.
The following examples are offered by way of illustration and not by way of limitation.
~PERIMENTAL
Example 1: High heparin concentration fetal bone marrow cultures.
Cells: K265 fetal bone marrow, 2x.
Media: IMDM + 5~ Gibco FCS + 10-5M 2-mercaptoethanol.
The FCS and 2-mercaptoethanol were added fresh each media change.
Heparin: Sigma 178 U/ng, stock solution 200 mg/ml.
Plate: 100 mm Falcon untreated plate.
Plate 1: No added heparin.
Plate 2: 1000 ~g/ml heparin sigma concentration.
Both plates were tended to by removal of 10 ml medium and addition of media plus heparin approximately every day, occasionally every other day. By day 10 a noticeable difference in the predominant cell morphology was evident.
Photomicrographs were taken evidencing the morphology;
Plate 1 had sheets of fibroblast-like cells which would form large balls of cells often dislodging from the plate.
Plate 2 had confluent layers of rounded cells with a different morphology. No sheets of fibroblasts were observed.

Example 2: High heparin concentration effect on fetal bone marrow cultures.
Bones: K266 and K267 samples were stripped of protein by cutting along the long axis in one half, incubating at 37 C with trypsin and every 0.5 hr, mixing for 3 hours followed by three washes with media. Cell concentration was 2X106 per ml.
Plates: 100 mm Falcon untreated plate.
Medium: IMDM plus 5% FCS plus lO-sM 2-ME.
Heparin: When used, 1000 ~g/ml (Sigma) diluted in sterile water.

~ 094/~306 215 0 4 7 1 PCT~S93/11870 Four plates were prepared, two each of K266 and K267, each pair including a plate with heparin and a control.
As observed in the previous experiment, the plates without heparin became overgrown in ten days to two weeks with elongated spindle-shaped cells. The plates with added heparin show the growth of the "egg-shaped" cells seen previously. These cultures were tended as previously described with change of media approximately QOD.

Example 3: The effect of different plates on meqakaryocyte and fibroblast growth.
The procedure described in Examples 1 and 2 was employed, using the cell preparation K275. Four plates were prepared, two tissue culture treated plates from Corning and two untreated plates from Falcon, where each pair included a control and a heparin-contAi~;ng medium.
The cultures were tended in the same way as previously described.
The tissue culture treated Corning control plate showed a rapid growth of adherent cells with a fibroblast morphology. Within 7 to 10 days, the culture was overgrown in areas and the cells peeled away from the plate. Often, balls of these cells were found floating in the medium. The medium became yellow rapidly despite frequent changes. In the tissue culture treated Corning plate with heparin-contA; n; ng medium, a mixed pattern of growth was observed with both fibroblast-like cells and cells with the "egg-shaped" morphology. The growth of the fibroblast-like cells was very significantly reduced and the overall cellularity of the culture was reduced compared to the control plate. At about 2-3 weeks, the fibroblast-like cells predominated in the culture and the - number of egg-shaped cells was reduced. Overall, the non-adherent cell number was minimal.
In the untreated Falcon plates, in the control plate, there was a rapid overgrowth of fibroblast-like cells.
These cells did not come to confluence as rapidly as in the tissue culture treated Corning control plate and there 2 Y5 ~ ~7 1 16 PCT~S93/11870 was a greater number of "egg-shaped" cells than in either of the tissue culture treated Corning plates. These cells form clusters in an area which seemed to exclude the fibroblast-like cells. Finally, in the untreated Falcon plate with heparin-contain~ng medium, growth to confluence of the egg-shaped cells was observed with eventual formation of multinucleated cells. An adherent layer of megakaryocytes, granulocyte-macrophages and stromal cells formed, supporting colonies with megablastoid cells.
Example 4: MethYlcellulose assay.
In the next study, a methylcellulose assay was carried out with conventional conditions, employing 20%
fetal calf serum and 100 ng/ml each of IL-l, -3 and -6 and erythropoietin. Whole bone marrow was employed at different seeding levels as well as selected cells based on CD34 and CD41. The CD34+ CD41+ is between 0.5 and 0.9%
of the whole fetal bone marrow population.
The following Table 1 indicates the results:

CELLS CELL COLONIES COLONIES CLUSTERS
WBM CD34 CD41 CONC. NO.* /loS
+ - - lxlO4ml 0-1-3 53 - + - lxlO4ml 1-2-0 40 - + + lxlO4ml10-7-19 480 4-5-7 - - - lxlO4ml 0-0-1 13 - - + lxlO4ml 0-1-0 13 + - - 2xlO4ml 0-1-5 40 2-3-2 - + - 2xlO4ml 1-2-1 23 1-1-1 - + + 2xlO4ml34-40-40 760 9-8-12 - - - 2xlO4ml 0-0-0 0 - - + 2xlO4ml 0-0-3 20 + - - lxlO5ml15-16-15 522 3-9-7 *These replicate clonies comprised 250 cells, while clusters comprise 20 cells.
Some colonies comprised morphologically identifiable megakaryocytes. Thus, colonies were observed at the lower concentration from the CD34+ CD41+ cells which matured to megakaryocytes. Later analysis has also shown that the colonies also contain cells in the granulocyte-macrophage lineage. In addition, when whole bone marrow was employed ~ 094/~306 2 1 5 ~ 4 7 1 PCT~S93111870 at a seeding of 105 per ml of cells, a significant number of colonies was observed which provided megakaryocytes. In addition, the CD34+ CD41+ population was found to be sensitive to the indicated factors, so that by removing one factor and replacing such factor with a test composition, one can determine the effect of such test factor on the formation of megakaryocytes.
A methylcellulose assay was done in the presence of IL-1, IL-3, IL-6 erythropoietin and FCS. The mean of 5 separate experiments gave the results:
Cell PopulationdaY 7 CFU-GM/lOs cells 34+41+ 602 34+41- 223 15 34-41+ 31 A methylcellulose assay was done in the presence of IL-3, Steel factor, erythropoietin and FCS with highly purified populations. The results show:
Cell PoPulation day 14 CFU-GM/lOs cells 34+41+ 4739 34+41- 1184 Example 5: Growth of meqakaryocYtes from buffy coat.
one unit of leukocytes (buffy coat) from an adult human contains about 2 x lo8 white cells. The red cells in the unit are lysed by a 10 min incubation at 37 C in a solution of ammonium chloride buffered with Tris-HCl. The leukocytes were then plated under stAn~Ard heparin culture conditions as described in Example 1 at 107 cells/10 cm plate, 5 x 106 cells/6 cm plate, or 105 cells/well in a microtiter plate in a volume of 10 ml, 5 ml, or 100 ~l, approximately 20 10 cm plates can be made.
After 2-3 weeks following the regimen described in Example 1, a confluent monolayer of megakaryocytes and precursors is obtained. ~he majority of other cell types are no longer present. If one assumes that the original inoculum contains 0.1~ megakaryocyte precursors, then this W094/~306 PCT~S93/11870 _ 2150~ 1 18 preparation has been expanded 103-fold, since a confluent 10 cm dish contains 107 cells.
Alternatively, aliquots of 1-2 x 107 fresh leukocytes can be frozen in liquid nitrogen in 10% DMSO and 20% FCS.
5 These vials can be thawed at a later time for use in r heparin cultures as described above.
ExamPle 6: MeqakaryocYte assaY by measurinq src kinase.
A. Immune complex auto kinase assay.
The cells emplo~ed were HL-60 (2 x 107) as a control, 10 platelets (1 ml) or heparin culture using human plasma.
The antibodies employed were mIgG (5 ~l) as a control, m-anti-src (EB8) (2.5 ~l), and m-anti-src (m--mouse) (2-17) .5 ~l).
The cells were lysed in 4 ml 1% NP40/1% deoxycholate lmM EDTA in buffer (150mM NaCl, lOmM NaPO4 buffer, pH 7.4, protease inhibitors) and immunoprecipitated as follows:
1.5 ml of each lysate was incubated overnight at 4 C with the indicated amount of antibody, followed by rotating at 4 C with 50 ~l of a 50% slurry of anti-mouse Ig coated Sepharose beads for 2 h. The beads were then washed 2x with 1 ml KLB buffer (1% Triton X-100, 10mM NaPO4 buffer, 150mM NaCl pH 7.4, and lx with 1 ml 20mM Hepes, pH 7, followed by dispersion in 40 ~l Hepes buffer, 5mM MnCl2, 5mM MgCl2, 15 ~Ci __32p ATP and incubated at 30 C for 20 min. After washing 3x with KLB + 0.05% SDS + 5mM EDTA, 20 ~l was loaded from beads resuspended in 35 ~l 2 x SDS
sample buffer (2% SDS 200mM Tris-HCl, pH 7.4, 5% 2ME, 20%
glycerol) onto a 10% polyacrylamide gel. The protein concentration of the lysates for immunoprecipitation (IP) was: HL-60 = 1.14 mg/ml; platelets = 0.86 mg/ml; heparin culture = l.lmg/ml. The gel was fixed in 5% acetic acid, 20% MeOH, the gel treated with lM KOH at 55 C for 2h, refixed, dried and exposed. No band in the gel was seen with HL-60, while a strong band was observed with both the heparin culture and platelets at a molecular weight position for src. CPM in the src protein band excised from the gel were 700 for the HL60 control, 21,000 for the heparin culture, and 68,000 for the platelets.

94/L~306 2 1 S 0 4 71 PCT/US93/11870 B. Src Kinase Elisa (Kiniza) This assay may serve as a megakaryopoiesis growth factor assay. In a 96 well plate, the wells are coated with anti-src, or control Ig. Cells are lysed with lysis 5 buffer (1% NP40, 10mM NaPO4 buffer, 150n~q NaCl, aprotinin, leupeptin) and the supernatants transferred to the antibody coated plate. After incubating for 3h at 4 C, the plates are washed 2x in KLB, lx with 20mM Hepes, and 50 ,lLl/well of 20TnM hepes, 5mM MnCl2/MgCl2, 1,uCi of _3~P-ATP
10 are added. After incubating at 30 C for 30 min, the plates are put back on ice and the reaction stopped with 100 ~1 NP40/DOC buffer (above). The plates are then washed 2-3x with the same buffer. The samples are eluted with 5%SDS at 75 C, the protein precipitated with carrier 15 protein added (FCS, ~ 1 ,lLl/well) in wells with 150 ,ul 20%
trichloroacetic acid. The precipitated protein is then transferred to 96 well dot blot apparatus with GF-C filter paper and washed with 10% TCA. The filter is cut and counted with a scintillation cocktail. Data is shown in 20 Table 2.

CPM
Lysis Buffer Ig 55 anti-src 104 Platelets Ig 2,998 (108/cc) Anti-src43,028 Heparin Culture Ig 631 Anti-src 3,520 Fetal bone marrow was sorted for the surface markers CD34+/- and CD41+/-. Cells were cultured in 96 well plates in the conditions described in Example 1. After an average of 4 weeks in culture the plates were assayed with the src kiniza assay.
The data below shows that the highest progenitor activity is found in the CD34+41+ population, as measured by src activity of the cells which grow out in culture.
The results are the mean of four experiments.

WO 94/L~306 PCT/US93/11870 2 1 ~ 20 Cell/well Population; CPM
105/well whole bone marrow 202 104/well CD34+, CD41- 76 5 104/well CD34~, CD41+ 684 104/well CD34-, CD41+ 329 104/well CD34-, CD41- 67 ExamPle 7: Isolation of meqakaryocyte growth factor.
10 5 g of heparin-Sepharose CL-6B (Pharmacia) were swollen in PBS to a final volume of 25 ml, followed by wA~h;ng in 1 liter of PBS. Fresh human plasma (25 ml) pooled from three donors was added to the swollen beads in a 50 ml tube and mixed at 4 C overnight. The plasma was 15 removed and filtered (referred to as "depleted"). Beads were then mixed for 5 h with 25 ml of IMDM containing 3 mg/ml heparin at 4 C. The medium was decanted and filtered (referred to as "eluate").
Heparin cultures were then set up as described in 20 Examples 1 and 6 with either bone marrow or buffy coat, where the various forms of plasma: (1) plasma (10%) used without treatment; (2) depleted plasma; or (3) depleted plasma plus the eluate (30% final volume), were used in place of the fetal calf serum described in Example 1. Src 25 kinase assay was performed on cultures grown 2 wks. Total protein in the dish was measured: CPM in src band was determined after excising band from the gel and scintillation counting. The results are shown in Table 4:

Lysate CPM in src ProteinSrc/
Source* band mq/ml 35 proteinl+
1) HL--60 60 0.80 1%
2) HEL 837 1.00 13%

3) HC, 10% HP4,860 0.73100%
40 4) HC, 10% HeSE316 0.25 19%
5) HC, 10% HeSE+F 3,0900.50 93%

094/~306 ~1~ 0 4 7 1 PCT~S93tll870 * HL-60 control line; myeloid leukemia HEL human erythroleukemia line HC heparin culture HP human plasma HeSE depleted plasma from heparin-Sepharose column F megakaryocyte growth factor eluted from heparin-Sepharose column (eluate) + Expressed as a percentage of culture #3 Example 8. Characterization of Adherent Cells from ~eqacultures Cultures were set up as previously described. After 12 days, adherent cells were removed by trypsin or EDTA, washed, and cytospun onto glass slides for immunostA;n;ng or megacolor staining. The results are shown in Table 5, comparing HL60, a myeloid cell line, megakaryocytes from bone marrow, and the megaculture adherent cells.

~ BM meqas Megaculture Cells Ig control Ib - + +
25 IIb - + +
src + + ++
Megacolor - + +
A "+" denotes that at least 10-50% of the cells stained positively for the marker. It can be seen from this data that the adherent cells from the megaculture stain for the cell surface markers which are characteristic of megakaryocytes.

Example 9. Megacolor Plate Assay Fetal bone marrow from sample J225 was sorted for the surface markers CD34+/- and CD41+/-. Cells were cultured in 96 well plates in the conditions described in Example 1.
The medium was removed prior to the assay, and the plates air dried. A fixative solution of 95% ethanol, 5% glacial acetic acid, 5% formaldehyde (37% solution) was added to each well, and then incubated at room temperature for 5 minutes. The fixative was removed, the wells washed 3 times with dH20, and air dried. 80 ~l per well of W094/~3~ PCT~S93/11870 ~
21S0~ 1 22 DNAse/RNAse solution (50 ml H20, 0.7 g sodium acetate, 1.23 g magnesium sulfate, 6.3 mg RNAse, 6.3 mg DNAse) was added to each well, which was then incubated at 37 for 30 minutes. The solution was removed, the wells washed 3 times with dH2O, and air dried. 80 ~1 of MegacolorTM
(Cytocolor) was added to each well, which was then incubated for 7 minutes at room temperature. The solution was removed, and the wells washed 4 times with H2O. 100 ~l of 2% SDS in PBS was added to each well, and then left for 1 hour to solubilize cells. Three triplicate wells were combined, and the absorbance read at ODs~. Table 6 shows the absorbance readings for the different sorted cell populations.

PRIMARY CULTURE
cells/well WBM34+41+ 34+41- 34-41+ 34-41-5 x 104 0 . 783 ND ND ND ND
20 2 x 104 0 . 3670.753 0.138 0.092 0.102 SECONDARY CULTURE
cells/well WBM34+41+ 34+41- 34-41+ 34-41-5 x 104 0.138 ND ND ND ND
2 X 104 0 . 0360.409 0.033 0.019 0.020 The data shows that the largest number of cells with megakaryocytic characteristics grow out of wells seeded with the CD34+CD41+ progenitor population. The number can be quantified by the Megacolor plate lysate assay.

Example 10: Aqar ColonY Assay Fetal bone marrow from sample K957 was sorted for the surface markers CD34+1- and CD41+/-. The cells were resuspended at 105/ml IMDM, 30% human plasma with the growth factors: 10 ng/ml human IL-3, 10-8 M PMA, and 10 ng/ml human GM-CSF. A sterile solution of 3~ bacto-agar was added to a final concentration of 0.3%. 1 ml of cells was plated in a 35mm non-tissue culture treated petri dish, and the agar allowed to set for 15 minutes at 4C.

~ 094/~3~ 2 1 5 ~ 4 7 1 PCT~Sg3/11870 The plates were placed in a large petri dish cont~; n; ng an open dish of sterile dH20, in a 37C incubator. After 13 days the granulocyte-macrophage colonies were counted. On day 14 or day 26, 33mm Whatman no. 1 filter paper was placed on top of the agar, the agar dried to a film, and the paper peeled off.
To assay for megakaryocyte colonies with Megacolor staining, the agar was fixed with a solution of 95%
ethanol, 5% glacial acetic acid, 5% formaldehyde (37%
solution) at room temperature for 5 minutes. The plates were washed 3 times with dH20, and air dried. DNAse/RNAse solution (50 ml H20, 0.7 g sodium acetate, 1.23 g magnesium sulfate, 6.3 mg RNAse, 6.3 mg DNAse) was added, incubated at 37 overnight, washed 3 times with dH2O, and air dried. Megacolor~ (Cytocolor) was added, the plates incubated for 7 minutes at room temperature, washed 5 times with dH2O and dried. The megakaryocytic colonies are stained purple.
The data in Table 7 shows that the CD34+41+
population has the highest frequency of megakaryocytic progenitors, and is also able to develop into granulocyte-macrophage colonies.

Population Colonies/10s cells Day 14 Day 14 Day 29 CFU-GMCD41+ colonies Meqacolor+
30 CD34+41+ 775 30 225 CD34+41- 128 5 o It can be seen from this data that the CD34+41+ population is enriched for granulocyte-macrophage progenitors, as well as for CFU-mega (day 14) and BFU-mega (day 26) cells.
For CD41 immunostaining of agar, 650 ~1 of CD41 (Serotec, purified and used at 1/300) was added to the agar plates, and incubated for 1 hour at room temperature.
The plates were washed with PBS 2 times, biotin conjugated horse anti-mouse IgG (used at 1/100) added, and incubated WOg4/~306 PCT~S93/11870 ~
2~5~4~1 24 for l hour at room temperature. Avidin conjugated alkaline phosphatase (Caltag, used at l/lO0) was added, the plates incubated for l hour at room temperature, and washed with PBS 2 times. The substrate of fast red Texas Red salt, naphthol AS-MX in propandiol buffer and levamisole was added, the plates washed with H2O, dried and examined for positive colonies.

ExamPle ll. Platelet production in vitro lO Megacultures were set up from buffy coat fraction in IMDM, with 5% human plasma and heparin. Supernatants from the cultures were harvested, and lOX CATCH medium added .
Catch medium is 1 mM theophylline, l.5% BSA, 1 mM EDTA, 0.8% ADC, pH 6.5 in Hank's BSS, where ADC is 93 mM
NaCitrate, 7 mM citric acid and 140 mM dextrose. Cells were spun out at llO0 rpm, lO minutes, and supernatant transferred to a new tube. Platelets were then spun out at 2500 rpm, lO minutes and resuspended in 40 ~l lX CATCH, and counted on a Baker automated cell counter. The data in Table 8 shows the number of platelets which can be harvested from the culture supernatant.

Expt. l Expt. 2 Day 7 Day 9 Platelets/ml untreated plate l.3 x 106 1 . 9 X 106 tissue culture 30 treated plate l. 4 X 105 4.l x lOs It can be seen from the above data that megacultures are able to provide for the production of platelets in vitro, and that production is enhanced in plates which have not been treated for tissue culture.

It is evident from the above results, that an efficient economic procedure has been provided for producing human megakaryocytes in culture. Thus, a continuous source of platelets is provided for treatment 21~0 171 W094/133~ PCT~S93/11870 and study of the function of platelets, as well as their formation. Also, megakaryopoiesis may be studied. In addition, a factor associated with megakaryopoiesis is provided and other factors may be similarly isolated.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.
All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims (20)

WHAT IS CLAIMED IS:

1. A method for producing megakaryopoiesis, said method comprising:
culturing CD34+ human hematopoietic progenitor cells or a megakaryocyte progenitor fraction thereof under megakaryocyte growth supporting conditions in a nutrient medium comprising at least one fibroblast growth inhibitin g agent; wherein said progenitor cells are characterized by the expression of at least one of CD34 and CD41.

2. A method according to Claim 1, wherein said medium stabilizes platelets.

3. A method according to Claim 2, wherein said medium comprises at least one of an ATP regenerating system, dextran acid citrate or low or substantially no calcium.

4. A method according to Claim 1, wherein said medium comprises a fibroblast inhibiting amount of heparin.

5. A method according to Claim 4, wherein said medium further comprises up to about 10% of human plasma.

6. A method for producing megakaryopoiesis, said method comprising:
culturing human hematopoietic progenitor cells or a megakaryocyte progenitor fraction thereof in a nutrient medium comprising about 5% human plasma, and from about 0-10 mM calcium;
wherein said culturing is in plastic or glass containers free of fibroblast growth supporting coatings;
and said progenitor cells are characterized by the expression of at least one of CD34 and CD41.

7. A method according to Claim 6, wherein said human hematopoietic progenitor cells are bone marrow cells.

8. A method according to Claim 6, wherein said human hematopoietic progenitor cells are derived from apheresed human blood.

9. A method for assaying for factors which regulate megakaryopoiesis, said method comprising:
seeding early hematopoietic progenitor cells in a liquid or semi-solid nutrient medium comprising plasma, said factor and at least one cytokine which is GM-CSF, IL-1, -3, -6 or erythropoietin; and determining the number of megakaryocytic cells which grow in greater than about 5 days as compared to the number of cells that grow under the same conditions in the absence of said factor.

10. A method according to Claim 9, wherein said semi-solid medium is selected from the group consisting of 0.8-1.5% methylcellulose and 0.2-0.5% agar; and said cytokine is present at a concentration of at least one-half of saturation of cellular receptors.

11. A conditioned medium comprising a nutrient medium for mammalian cells, at least one agent for inhibiting fibroblast formation, secreted proteins of adherent cells grown from bone marrow and capable of supporting megakaryopoiesis.

12. A method for treating a patient requiring platelets, said method comprising:
infusing into said patient a cellular composition comprising at least 70% CD34+41+ human cells, said human cells being characterized by the ability to give rise to megakaryocytes.

13. A method according to Claim 13, wherein said composition comprises from about 106 - 108 of said human cells.

14. A method according to claim 13, wherein said cellular composition is derived from apheresed adult human blood.

15. A cellular composition comprising a major member of human megakaryocytic progenitor cells, where said progenitor cells are characterized as CD34+, CD41+ and having the ability to initiate megakaryocytic cultures n vitro;
said cultures giving rise to cells which stain positively with Megacolor, giving rise to cells solely in the myelomonocytic lineage, are CD41+CD34- and give a positive signal in a src kinase assay.

16. A composition according to claim 15, wherein at least 70% by weight of the cells are megakaryocytic progenitors.

17. A method according to claim 16, wherein said cellular composition is derived from apheresed human blood.

18. A cellular composition comprising a major member of human hematopoietic progenitor cells, where said progenitor cells are characterized as CD34+, CD41+ have the ability to initiate mixed cultures in vitro, said mixed cultures comprising cells of the megakaryocytic, granulocytic and macrophage lineages.

19. A composition according to claim 18, wherein at least 70% by weight of the hematopoietic progenitor cells are CD34+41+.

20. A composition according to claim 19, wherein said cellular composition is derived from apheresed human blood.